Dopamine neurons are a critical component of reward pathways in the brain. Historically, dopamine neurons were considered a relatively homogeneous population mediating association of reinforcement signals from food, water, and reproduction with coinciding sensory stimuli. However, recent studies have shown that these neurons form subpopulations with distinct physiological profiles, neural projections, and biological functions. These distinct subpopulations also have different roles in the progression of the addiction cycle, from use to abuse, abstinence and relapse. The goal of this application is to engineer novel genetic tools that will allow precise manipulation of distinct subpopulations of dopamine circuits, with specificity down to single pairs of neurons. To this end we will first apply a new technique, single-cell ATAC-seq, to determine all open chromatin/accessible DNA enhancer elements of every one of the ~250 dopamine neurons in the Drosophila brain. Second, we will determine which open enhancer fragments, or combinations thereof, will uniquely identify single dopamine cells/cell-types. And third, based on this analysis, we will engineer numerous new genetic tools and test them for their in vivo efficacy and specificity. While these Aims are linear and fully interdependent, the grant overall applies the very new technique of single-cell ATAC-seq to ?break new ground? and ?accelerate the pace of discoveries to advance addiction research?. The proposal essentially tests the hypothesis that this unbiased, genome-wide approach can be harnessed to generate new tools for precision intervention. Because the approach itself can be scaled, applied to any cell type, and translated to mammals, this application is fully consistent with the spirit of the Cutting-Edge Basic Research Awards (CEBRA) mechanism, which will ?support high-risk, high impact research? (PAR-18-437).